Kasper, T., et al. (1988). Separation and Detection of DNA by Capillary Electrophoresis. Journal of Chromatography. 458, 303-312 DOI Link
Introduction
- The physical properties of fused silica capillaries and the variety of separation strategies that may be imployed in capillary electrophoresis make it an ideal medium for electrophoresis in free solution.
-Traditional techniques such as zone electrophoresis and micellar electropheresis fail to separate large DNA molecules effectively, since they have effectively the same size to charge ratio.
-This group designed separations of oligonucleotides and restriction fragments up to 23kb in size.
Materials and Methods
- The authors describe the source of all nucleotides and solvents used, as well as the source of the fused-silica capillary.
- The authors describe the brand and model of the instrument and detector utitlized, as well as modifications to the capillary.
- Experimental conditions for the separation procedure and detection are described.
Results and Discussion
- Concentrations of DNA was detected over 3 orders of magnitude
- DNA stained with EB dye shows greatest sensitivity at low DNA concentrations, unlike traditional gel staining which shows a linear response over a wide range of DNA concentrations.
- Absorbance detection of DNA is more sensitive and shows a larger linear response, but stained DNA reduces background noise and eleminates certain artifacts.
- MECC can effectively separate nucleotide phosphates and oligonucleotides, as well as basic, acidic, and uncharged peptides.
- Oligonucleotides with sizes between 4 and 22 bases can be separated by CE with the addition of 5% ethylene glycol.
- The tertiary structure of different DNA molecules causes large differences in the Stokes radius and therefore their viscous drag, resulting in their separation.
- Specific complexation can be used to separate DNA fragments using a CTAB-micelle buffer, which acts as an ion-pairing agent.
- Specific complexation shows the largest number of theoretical plates for the separation (~1,000,000), and warrants further investigation.
Conculsion
-CE can be used to separate large DNA molecules, although the mechanisms are poorly understood.
- It is assumed these same methodologies can be applied to RNA separations as well.
Kasper, T., et al. (1988). Separation and Detection of DNA by Capillary Electrophoresis. Journal of Chromatography. 458, 303-312 DOI Link
Introduction
- The physical properties of fused silica capillaries and the variety of separation strategies that may be imployed in capillary electrophoresis make it an ideal medium for electrophoresis in free solution.
-Traditional techniques such as zone electrophoresis and micellar electropheresis fail to separate large DNA molecules effectively, since they have effectively the same size to charge ratio.
-This group designed separations of oligonucleotides and restriction fragments up to 23kb in size.
Materials and Methods
- The authors describe the source of all nucleotides and solvents used, as well as the source of the fused-silica capillary.
- The authors describe the brand and model of the instrument and detector utitlized, as well as modifications to the capillary.
- Experimental conditions for the separation procedure and detection are described.
Results and Discussion
- Concentrations of DNA was detected over 3 orders of magnitude
- DNA stained with EB dye shows greatest sensitivity at low DNA concentrations, unlike traditional gel staining which shows a linear response over a wide range of DNA concentrations.
- Absorbance detection of DNA is more sensitive and shows a larger linear response, but stained DNA reduces background noise and eleminates certain artifacts.
- MECC can effectively separate nucleotide phosphates and oligonucleotides, as well as basic, acidic, and uncharged peptides.
- Oligonucleotides with sizes between 4 and 22 bases can be separated by CE with the addition of 5% ethylene glycol.
- The tertiary structure of different DNA molecules causes large differences in the Stokes radius and therefore their viscous drag, resulting in their separation.
- Specific complexation can be used to separate DNA fragments using a CTAB-micelle buffer, which acts as an ion-pairing agent.
- Specific complexation shows the largest number of theoretical plates for the separation (~1,000,000), and warrants further investigation.
Conculsion
-CE can be used to separate large DNA molecules, although the mechanisms are poorly understood.
- It is assumed these same methodologies can be applied to RNA separations as well.